The present invention relates to a recombinant bacterium based on a non-pathogenic bacterium that has a modified genome containing a nucleic acid of interest from a pathogen that is detected by a molecular diagnostic assay and that mimics the diagnostic profile of the pathogen. The invention further relates to a diagnostic control composition comprising the recombinant bacterium and to methods for producing the recombinant bacterium. The recombinant bacterium is a safe, reliable quality control for the detection of pathogens such as Mycobacterium tuberculosis and Staphylococcus aureus. 
Tuberculosis (TB) is a devastating disease that is recalcitrant to effective clinical management. Its robust nature combined with outdated diagnostic methods have made treatment at the point of care increasingly difficult, especially in the ever-increasing prevalence of drug resistant strains. With diagnostics in many microbial diseases shifting away from bacteriologic culture and microscopic identification methods towards molecular biological systems, automation requires stringent calibration to ensure sensitivity and specificity of the results. The World Health Organisation (WHO) has endorsed two nucleotide amplification assay (NAA) diagnostic tests for standard identification of Mycobacterium tuberculosis in pulmonary patients that can simultaneously identify the organism as well as assess RIF and/or INH resistance, namely GeneXpert MTB/RIF (Cepheid Innovation) and the GenoType MDRTBplus Line Probe Assay (LPA) (Hain Lifescience®). This has empowered clinicians in tailoring drug regimens for patients, thereby not only improving treatment outcomes but also restricting spread in communities. Here we report modified strains of the related non-pathogenic soil bacterium Mycobacterium smegmatis that mimic the diagnostic profile of H37Rv in both the GeneXpert® MTB/RIF (Xpert® MTB/RIF) and the Hain-Lifescience® LPA, in terms of both organism identification and RIF resistance profiling. To extend this approach to other diseases we also show that a Staphylococcus aureus gene sequence can be introduced into Mycobacterium smegmatis to generate a positive response in the GeneXpert® SA Nasal complete (Xpert®-SANC) cartridge, designed for identification of methicillin resistance in Staphylococcus aureus. This finding demonstrates that this application holds promise across diagnostic platforms for production of a surrogate positive NAA signal from a non-infectious organism. Currently the calibration standards for the Xpert® MTBIRIF assay are produced in a laboratory strain of Mycobacterium tuberculosis H37Rv, the procedure of which is lengthy, hazardous and costly as it requires mass production of live bacteria which are then killed to make them safe to handle and transport. The modified Mycobacterium smegmatis holds promise as a replacement for large scale calibration standard production, external quality assessment and general clinical surveillance as worldwide demand of these diagnostics is expected to increase.
The current regimen for TB treatment prescribed by the WHO consists of four drugs: isoniazid (INH), pyrazinamide (PZA), ethambutol (EMB) and rifampicin (RIF)). RIF is potent at killing Mycobacterium tuberculosis through binding of the RNA polymerase (RNAP) beta subunit RpoB of the transcription complex, thus inhibiting the synthesis of RNA and consequently proteins. Resistance develops by non-synonymous amino acid substitutions that prevent the binding of RIF to the active site of RNAP, whilst retaining functionality of the complex. In the presence of RIF, in the clinic and the laboratory, sensitive strains are selected against, leading to an outgrowth of drug resistant mutant strains. Whilst mutation to RIF resistance (RIFR) carries a fitness cost under normal growth conditions, these resistant strains have ensured their own survival by their chromosomally encoded adaptation under conditions in which all other RIF susceptible (RIFS) strains die.
The majority of RIFR strains have single nucleotide polymorphism (SNP) mutations confined to a limited sequence within the rpoB gene, the 81 bp RIF resistance determining region (RRDR). As one of the first line anti-tubercular drugs, the efficacy and low cost of RIF is critical for continued use against drug sensitive clinical strains. Prevention of morbidity and mortality is compromised by the treatment of patients infected with resistant strains which are non-responsive to antibiotics in the regimen. In addition, these patients contribute to spread of resistant bacteria in communities. In order for clinicians to make informed decisions, smear microscopy is no longer sufficient. Ideally drug susceptibility testing (DST) needs to be performed alongside identification of the causative agent Mycobacterium tuberculosis. 
Strains that are multidrug resistant (MDR), i.e. no longer responsive to RIF and INH, are effectively treated with only EMB and PZA, and in the process are more likely to evolve further resistance to these drugs. It is then necessary to switch to alternate drug regimens to ensure continued efficacy. Drug therapy to combat MDR-TB is significantly more expensive, requiring at least 5 different drugs from an array consisting of fluoroquinolones (levofloxacin or moxifloxacin), injectable drugs (amikacin, kanamycin or capreomycin), oral bacteriostatic drugs (prothionamide, ethionamide, cycloserine or para-aminosalicylic acid) and pyrazinamide for up to 2 years. Patient compliance is a major risk factor, especially for the injectable drugs which require clinical administration.
Due to the slow growth of Mycobacterium tuberculosis in vitro, lengthy culture times are required to obtain growth of bacteria for identification, followed by additional culture in the presence or absence of antibiotic to test for resistance. The current gold standard for growth assessment by liquid culture is the BACTEC™ MGIT™ (mycobacterial growth indicator tubes) in which a test is considered negative only after 42 days. If at any time before that bacteria emerge, this test is considered positive. As a result, it can again take 42 days to make a decision regarding infection and a further 42 days more to determine the drug resistance of the organism. This can lead to patients being treated with ineffective antibiotics in the interim, during which time their health will deteriorate and the strain will potentially spread.
Recent developments in molecular diagnostic methods have significantly improved upon culture methods, as they probe the sputum sample directly for the presence of the bacterial chromosome. It is for this reason that in 2010 the WHO endorsed two nucleic acid amplification (NAA) diagnostic assays for TB. The GeneXpert® real time PCR platform has been developed for use with a number of different assays. The Xpert® MTB/RIF assay utilises molecular beacon technology that allows for real time signal produced by NAA. As the amount of nucleic acid increases with every round of amplification, more fluorescent signal is released and highly specific sequence can be detected. The test is entirely automated and requires little instruction or skill of clinical staff, wither for operation or evaluation of results.
The Xpert®-MTB/RIF system relies on a fixed set of gene probes that can specifically identify the Mycobacterium tuberculosis-specific sequence and differentiate it from other bacteria that differ marginally, including non-tuberculous mycobacteria and related soil bacteria. These differences, although discernible at the molecular level, are non-synonymous and do not affect the peptide sequence of the gene nor the function of the resulting RpoB protein. The same principle allows GeneXpert® to identify Mycobacterium tuberculosis-specific sequence which does not match the wild type sequence at different positions. In this case however the peptide sequence is altered leading to mutant proteins. This is the cause of the phenotype that results in antibiotic resistance.
The principle behind the molecular line probe assay (LPA) (Genotype® MTBDR plus assay, HAIN Lifescience®, Germany) differs in that it generates information about the presence or absence of nucleic acid content related to drug resistance at the endpoint of the amplification procedure. Several steps of processing are involved and each requires a number of parallel controls, and the procedure requires technically skilled personnel.
Staphylococcus aureus is a gram-positive, round-shaped bacterium frequently found in the nose, respiratory tract, and on the skin. Although Staphylococcus aureus is not always pathogenic, it is a common cause of skin infections such as a skin abscess, respiratory infections such as sinusitis, and food poisoning. Pathogenic strains often promote infections by producing virulence factors such as potent protein toxins, and the expression of cell-surface proteins that bind and inactivate antibodies.
The emergence of antibiotic-resistant strains of Staphylococcus aureus such as methicillin-resistant Staphylococcus aureus (MRSA) is a worldwide problem in clinical medicine. MRSA is believed to have evolved by acquiring a mobile genetic element, the Staphylococcal cassette chromosome (SCC) by horizontal transfer from another species. The SCCmec cassette carries the mecA gene which confers methicillin resistance. Despite much research and development there is no approved vaccine for Staphylococcus aureus. Over 278,000 hospitalized persons are infected by MRSA annually and MRSA accounts for over 60% of hospital-acquired Staphylococcus aureus infections in the United States. MRSA strains are particularly virulent, spread rapidly and cause more severe infections than other Staphylococcal bacteria. Early detection of MRSA and the ability to distinguish MRSA from methicillin-sensitive Staphylococcus aureus assists in limiting the spread of infection, determining treatment options and reducing healthcare burden. It has been shown that active surveillance of MRSA infections optimises effectiveness and control programs from MRSA outbreaks.
The GeneXpert® MRSA assay (Xpert®-MRSA) targets DNA sequences in the region of the open reading frame orfX where the staphylococcal cassette chromosome mec (SCCmec) integrates into the Staphylococcus aureus chromosome. SCCmec carries the resistance determinant mecA, which encodes methicillin resistance and exhibits at least six different structural types and numerous subtypes.
The Xpert®-MRSA and Xpert®-SANC are widely used in active surveillance of MRSA. However, there is presently no calibration or surveillance standard for testing the accuracy of the test as applied to surveillance testing in order to reduce false positive and negative results.
The use of live strains of bacteria as positive controls in diagnostic testing is undesirable for virulent bacteria as they pose a risk for the operator in ordinary handling in the laboratory. Killing these bacteria to produce a positive control requires the use of specialised laboratories with skilled staff.
In this work we have generated strains of the soil bacterium Mycobacterium smegmatis, a non-pathogenic relative of Mycobacterium tuberculosis, to test whether it is possible to utilise these as verification standards for diagnostic procedures. GeneXpert® assays and the Hain Lifescience® LPA require controls that confirm the accuracy of the assays. The Mycobacterium smegmatis strains reported herein contain nucleotide sequences introduced from either Mycobacterium tuberculosis or Staphylococcus aureus that mimic the diagnostic profile of clinical strains. These strains are expected to significantly reduce the cost, time and biohazard risk in the production of verification standards, with possible applicability across other NAA platforms.